Repair of UV-induced DNA damage is central to the prevention of a number of adverse conditions such as melanoma, but in most cases the mechanism of such DNA repair is not well understood at a molecular level. Under normal cellular conditions, the major DNA photoproducts of UV irradiation are cyclobutane pyrimidine dimers (TT, CT, and CC dimers) and 6,4-photoproducts. 5-Thyminyl-5,6-dihydrothymine is typically a minor product of UV irradiation. This typically minor UV photoproduct becomes the major UV photoproduct under certain conditions, however, including the conditions that exist in bacterial spores (thus the common name is spore photoproduct, SP). Remarkably, the formation of spore photoproduct is correlated with the unusually high resistance of bacterial spores to UV irradiation, and this resistance arises in part from the novel DNA repair enzyme that repairs SP. The overall goals of this proposal include investigating the mechanism by which the repair enzyme, SP lyase, recognizes and repairs SP. In addition, experiments designed to probe the mechanism by which SP is formed at the expense of cyclobutane pyrimidine dimers, are described. The specific aims of this proposal are as follows: 1) To investigate the structure and environment of the iron-sulfur cluster in spore photoproduct lyase using appropriate spectroscopic methods; 2) To probe the mechanistic details of the DNA repair reaction using isotope labeling and substrate analogs; 3) To investigate the molecular basis of the interaction of SPL with UV-damaged DNA using gel-shift assays and DNA footprinting; 4) To investigate the interaction between the iron-sulfur cluster of SP lyase and its substrates S-adenosylmethionine and spore photoproduct using Mossbauer, electron-nuclear double resonance, and electron-spin echo envelope modulation; and 5) To investigate the structural basis of SP formation using biochemical and X-ray structural methods. [unreadable] [unreadable]